are cells prone to swelling in PBS after sorting - (Jul/21/2009 )

I noticed quite some times that the size of the cell pellet I collected after sorting got shrank (significantly) after I washed the cells with PBS. Does anybody else has the same issue? Is there any way to avoid the loss of cells? Thanks a bunch!

The cells I am collecting are really precious and it is painful to see that happening.

-TracyDuke-

TracyDuke on Jul 21 2009, 09:46 AM said:

I noticed quite some times that the size of the cell pellet I collected after sorting got shrank (significantly) after I washed the cells with PBS. Does anybody else has the same issue? Is there any way to avoid the loss of cells? Thanks a bunch!

The cells I am collecting are really precious and it is painful to see that happening.

Cells should not shrink in PBS, so you may want to use another lot of PBS, just in case if has gotten concentrated after evaporation.

Other reasons:

You sure that it is not due to reduced number of cells after sorting?

Is your centrifugation speed right?

I would just count the number of cells before and after washing, and get a look at their size too.

-cellcounter-

Thank you Cellcounter!
I tried comparing by counting the cells before and after washing. And I found that the number of the cells dropped to about 1/5!! I was shocked. Is it possible that because the cells go through some sort of presure from the FACS machine which makes the cells fragile and therefore easily swell/burst in PBS?

-TracyDuke-

TracyDuke on Jul 25 2009, 12:20 AM said:

Thank you Cellcounter!
I tried comparing by counting the cells before and after washing. And I found that the number of the cells dropped to about 1/5!! I was shocked. Is it possible that because the cells go through some sort of presure from the FACS machine which makes the cells fragile and therefore easily swell/burst in PBS?

Probably not. Check the supernatant after centrifugation, I think you may find your "lost" cells there.

Anyway, cells do not like being in PBS (or any other protein-free solution) for too long. Do you sort cells into tubes containing some medium/protein? I find that tissue culture medium (bicarbonate-free, pHed to 7.2) containing 5% FCS is optimal for cell collection and washing. If you need serum-free, medium plus 0.1% BSA should be fine.

You need bicarbonate-free medium so you donīt get pH changes in the absence of CO2 gassing.